Gene transfer in poultry by introduction of embryo cells in ovo

ABSTRACT

A method of altering the phenotype of a bird comprises introducing a DNA sequence into somatic cells of a bird contained within an egg during in ovo incubation. The DNA sequence is selected to be effective to cause a change in phenotype, such as an increase in growth rate, feed efficiency, or both in the bird after hatch. A DNA sequence may further be selected to increase disease resistance or induce disease prevention by the expression of an antigen over a period of time.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 07/826,030, filed 27 Jan. 1992, the content ofwhich is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to the methods of altering the phenotypeof birds by introducing avian embryo cells into an egg containing thebird prior to hatch, which embryo cells carry heterogenous geneticmaterial.

BACKGROUND OF THE INVENTION

[0003] Commercial poultry is an extremely important source of food.However, there has been comparatively little attention given to methodsof producing useful changes in the phenotype of birds through geneticengineering techniques. This is unfortunate, because such techniquesoffer a much more rapid technique for introducing desirable phenotypictraits into birds than classical breeding techniques.

[0004] Currently, the most widely investigated method of genetransfection in poultry employs retroviral vectors. Exemplary is Souzaet al., J. Exptl. Zool. 232, 465-473 (1984), in which a retroviralvector encoding growth hormone was injected into the vascularizedportion of the yolk sac of 9 day old embryos. See also Shuman andShoffer, Poult. Sci. 65, 1437-1444 (1986); Salter et al., Poultry Sci.65, 1445-1468 (1986); Salter et al., Virology 157, 236-240 (1987);Bosselman et al., Science 243, 533-535 (1989); and U.S. Pat. No.5,162,215 to Bosselman et al.

[0005] Nabel et al., Science 249 1285-1288 (1990), and Wolff et al.,Science 247, 1445-1468 (1990), state that transient expression of 2-5months may be obtained from direct microinjection of DNA, but do notsuggest how these techniques may be applied to genetically engineeringpoultry. Nabel et al. note that the expression of DNA encodingβ-galactosidase injected into porcine arterial segments was limited tothe microinjection site. Acsadi et al., New Biologist 3, 71-81 (1991)state that myocardial cells were able to transiently express injectedforeign genes.

[0006] Simkiss et al., Protoplasma 151, 164-166 (1989) indicate thatprimordial germ cells of Stage XVII embryos containing endogenousretroviral sequences can be transferred to comparable recipient StageXVI embryos that lack the retroviral marker by cardiac puncture. At day17 of incubation, dot blots on recipient birds showed donor DNA to bepresent in the gonads, and traces of donor DNA to be present in theliver and heart tissues. The expression of the injected DNA moleculeswas not reported.

[0007] PCT Patent Application Serial No. US90/01515 discloses a methodof delivering a nucleic acid sequence to the interior of a vertebratecell. Injection of a DNA molecule into poultry was not reported.

[0008] In view of the foregoing, an object of the present invention isto provide methods of changing the phenotype of birds through geneticengineering procedures.

[0009] An additional object of the present invention is to provide amethod of changing the phenotype of birds in which expression of anexogenous DNA sequence is sufficient produce the phenotypic change.

[0010] Another object of the present invention is to provide a method ofchanging the phenotype of birds which is rapid and convenient.

SUMMARY OF THE INVENTION

[0011] A first aspect of the present invention is a method of alteringthe phenotype of a bird. The method comprises introducing a DNA sequenceinto somatic cells of a bird contained within an egg during in ovoincubation, with the DNA sequence being effective to cause a change inphenotype in said bird after hatch (e.g., a change in growth rate, feedefficiency, disease resistance, or a combination of all of thesefactors). Introduction of the DNA may be carried out by any suitablemeans, including injecting the DNA sequence in ovo into any compartmentof the egg including the body of the embryo. Preferably, the egg intowhich the DNA is introduced is incubated to hatch, and the bird soproduced raised to at least an age at which the change in phenotype isexpressed.

[0012] In an illustrative embodiment of the foregoing, the DNA sequenceis introduced by first transfecting avian hematopoietic progenitor cellswith the DNA sequence in vitro, and then injecting said transfectedhematopoietic progenitor cells into the egg, preferably into the yolksac or onto the chorioallantoic membrane, and preferably during earlyembryonic development.

[0013] A second aspect of the present invention is a method of alteringthe phenotype of a bird comprising introducing avian somatictissue-specific stem cells to an egg containing a bird during in ovoincubation, wherein the avian somatic tissue-specific stem cells containand are capable of expressing at least one DNA molecule in an amounteffective to cause a change in the phenotype of the bird. Introductionof hematopoietic stem cells is a preferred embodiment, as it has beendemonstrated that a DNA molecule contained therein persists and canexpress the protein encoded therefor at the introduction site, the bonemarrow, and in the peripheral blood of the embryo. It is also preferredthat the somatic tissue-specific stem cell be introduced to the birdduring a developmental stage at which the cells responsible for thephenotypic expression desired to be altered are colonizing within thetarget tissue.

[0014] A third aspect of the present invention is a method of alteringthe phenotype of a bird comprising introducing avian embryo cells to theair cell of an egg containing a bird during in ovo incubation, whereinthe avian progenitor cells contain and are capable of expressing atleast one DNA molecule in an amount effective to cause a change in thephenotype of the bird. The inventors have demonstrated that embryo cellsintroduced into the air cell migrate across the air cell membrane andcolonize the appropriate target tissue. Preferred cell types areembryonic stem cells and primordial germ cells.

[0015] A fourth aspect of the present invention is a method of alteringthe phenotype of a bird comprising introducing avian somatictissue-specific stem cells to the air cell of an egg containing a birdduring in ovo incubation, wherein the avian somatic tissue-specific stemcells containing and capable of expressing at least one DNA molecule inan amount effective to cause a change in the phenotype of the bird.Preferred cells for introduction are hematopoietic stem cells and neuralcrest stem cells.

[0016] A fifth aspect of the present invention is a bird produced by theforegoing methods.

[0017] A sixth aspect of the present invention is the use of a DNAsequence for the preparation of a medicament for producing a phenotypicchange in a bird by introducing the medicament in ovo, as describedabove.

[0018] A seventh aspect of the present invention is an apparatus for theintroduction of a DNA sequence in an egg during in ovo incubation, theDNA sequence capable of producing a phenotypic change in the birdcarried by the egg after hatch, as described above.

DETAILED DESCRIPTION OF THE INVENTION

[0019] There are several aspects of avian embryonic development whichmake it an attractive target for DNA introduction by stem cell transfer.First, since the greatest period of embryonic development occurs in theegg outside the maternal reproductive tract, the embryo can be easilyaccessed for the introduction of exogenous DNA.

[0020] Second, the fact that the egg is a multi-compartmentalized unitcan be exploited to deliver biological materials to specific embryonicsites. For example, the yolk sac in the early embryo functions tomanufacture blood. Immediately prior to hatching, the yolk sac serves aprimarily nutritional function and is taken into the intestinal tractand thereby transported to the cecal pouches during and after hatch.Therefore, yolk sac administration of materials can lead to bothembryonic cecal or vascular system delivery. Vascular system deliverythrough administration of DNA into the yolk sac would be particularlydesirable for administering DNA constructs capable of expressingphysiologically active peptides in the bird, such as growth hormone,lymphokines such as interferon and interleukin-2, insulin-like growthfactor, thyroid releasing hormone (TRH) or epidermal growth factor. Inaddition, administration of a peptide or DNA construct can beefficiently carried out by injection of the molecule onto thechorio-allantoic membrane or onto the air cell membrane. Finally, accessto the embryonic musculature compartment can be achieved by directembryonic injection at transfer in the last quarter of incubation, andin chickens, preferably in days 17-19 of incubation.

[0021] Third, there are several poultry embryonic cell lineages, such asblastodermal and germ cells, hematopoietic stem cells derived from aortaand yolk sac, and neural crest cells, that can be used as cellularvehicles for gene targeting.

[0022] Fourth, it is of no deleterious consequence if the transformedembryo and chicken is chimeric, so long as a physiological response isachieved in the animal after hatch sufficient to evoke the phenotypicchange sought.

[0023] The foregoing and other aspects of the present invention areexplained in greater detail below.

[0024] A. Phenotypic Alteration

[0025] The present invention provides a number of methods of alteringthe phenotype of a bird after hatch by in ovo introduction of a DNAmolecule contained within a somatic stem cell or primordial germ cell tothe bird. As used herein, an altered “phenotype” of a bird is intendedto encompass a sustained alteration in the cellular biochemistry of abird by the expression of a foreign DNA molecule within the tissues ofthe bird, which alteration results in a change in one or more physicalcharacteristics of the bird. Thus an altered phenotype can be a changein size, appearance, endocrine response growth rate, immune response tospecific antigens, metabolic rate, feed consumption and efficiency,gender, and the like. Alternatively stated, the present inventionprovides methods for inducing a physiological response (e.g., an immuneresponse, or a hormonal or endocrine response) in a bird after hatchthrough administering to a bird in ovo a DNA molecule encoding andexpressing a protein or peptide, which DNA molecule is administered inan amount effective to induce said physiological response after hatch.Note that the physiological response may be directly induced afterhatch, or may be indirectly induced after hatch (such as by induction ofa physiological response prior to hatch which endures after hatch), orbe a constitutive expression initiated prior to hatch.

[0026] A particular altered phenotype of interest is a change in immuneresponse wherein introduction of an avian embryo cell containing a DNAmolecule immunizes the bird. Exemplary DNA molecules for introductionare those that encode a protective antigenic protein or peptide thatinduces an immune response from the recipient bird. This can be done incombination with or in lieu of vaccination of the bird to protectagainst a specific pathogen.

[0027] Altering the endoqenous immune response of a bird in ovo is ofparticular interest due to the presence of maternal antibodies inembryonic and young mammals and birds. Maternal antibodies can interferewith typical vaccination programs for these animals. These antibodies,provided to the neonate from the bloodstream of the mother, conjugatewith specific antigens and thus provide natural protection against thoseantigens prior to the development of immunocompetence by the neonate.Unfortunately, maternal antibodies can also hinder typical vaccinationprotocols; they bind to the immunogenic component of the vaccine andthus inhibit neonatal production of antibodies. The presence of maternalantibodies precludes vaccination early in the development of theneonate. Typically, multiple vaccination protocols are required so thatactive immunization can occur once maternal antibody levels havedecreased to a sufficiently low level that they will no longer interferewith the vaccine.

[0028] A novel strategy for counteracting maternal antibody interferencewith vaccination is also disclosed herein. One aspect of this inventionis a method of immunizing a bird comprising introducing a DNA moleculethat encodes an antigen into the muscle tissue of a bird containedwithin an egg in ovo in an amount sufficient to neutralize maternalantibodies. Once neutralized, the maternal antibodies no longerinterfere with a vaccine containing the antigen; thus such a vaccine canbe used to immunize the bird. Alternatively, the DNA molecule can beintroduced in an amount effective so that, upon expression, not onlydoes the antigen neutralize maternal antibodies, but also provides animmunogen which vaccinates the bird against a specific pathogen.

[0029] The DNA molecule introduced can be any molecule that encodes anantigen that will neutralize maternal antibodies present in the bird.Exemplary antigens of interest include those produced by Marek's,infectious bronchitis, mycoplasma, avian leucosis, reovirus, pox,adenovirus, cryptosporidia, chicken anemia agent, Pasteurella species,avian influenza, Marek's MDX virus, Gumboro Disease virus, NewcastleDisease Virus (NDV), Infectious Bursal Disease Virus (IBDV), Roussarcoma virus, Escherichia coli, and Eimeria species such as Eimeriatenella (causing coccidiosis).

[0030] The cell can be introduced by any of the methods set forth below,and can contain any of the DNA construct configurations set forth below.

[0031] It is preferred that the DNA molecule be introduced so that theantigen is expressed as or after the embryo develops immunocompetence,more preferably in the last quarter of incubation. At immunocompetence,surface antigens encoded by the DNA construct can stimulate both a B-and T-cell response, resulting in immunization before challenge bypathogens encountered in the field after hatch. The timing and theduration of the last quarter of incubation varies among different avianspecies due to the variation in incubation duration. For example, forchickens, the last quarter of incubation is from about day 16 to hatch;for turkeys, the last quarter is from about day 19 to hatch.

[0032] Other altered phenotypes of particular interest includemodification of size, growth rate, feed efficiency, metabolic rate,endocrine response, neural system structure and function, and gender.

[0033] B. Gene Targeting

[0034] 1. Stem Cell Injection

[0035] As used herein, the term “embryonic stem cell” is intended torefer to embryonic cells that are uncommitted to any differentiationpath, or “totipotent”; i.e., their ultimate function in the mature birdis undetermined, as they can differentiate along any cell lineagepathway and terminally differentiate into any mature cell type. It isgenerally believed that embryonic stem cells exist in the embryo up tothe developmental stage at which the embryo, still a blastula, comprisesbetween about 8 and 64 cells. The term “tissue-specific stem cell”refers to an embryonic cell which is developmentally committed to aparticular tissue type, but which can still differentiate into one of aplurality of cell types within the tissue (i.e., are “pluripotent”), andwhich retain the ability to self-renew. Exemplary tissue-specific stemcells are primoridal germ cells and somatic stem cells; somatic stemcells include, but are not limited to, hematopoietic stem cells, whichdifferentiate to form the mature cells of the lymphocytic and myelocyticlineages, and neural crest stem cells, which differentiate to formportions of the nervous system and melanocytes. The term“non-tissue-specific stem cells” refers to self-renewing cells which areno longer totipotent, but which nonetheless are not committed to aspecific tissue type. Examples of these cells include the cellscomprising the ectoderm, endoderm, and mesoderm of the embryo. The term“embryo cell” is intended to encompass embryonic stem cells, non-tissuespecific stem cells, and tissue-specific stem cells as defined above.Avian embryo cells (e.g., chicken, turkey, duck, goose, quail, pheasant)are preferred, and it is particularly preferred that the embryo cells beof the same species as the egg into which they are introduced. However,it is contemplated that non-avian cells (e.g., reptile, mammalian, suchas bovine, ovine, procine, or murine) may also be employed.

[0036] The present invention encompasses the introduction of avianembryo cells into a bird in ovo to alter the phenotype of the bird. Inone embodiment, it is preferred that the avian embryo cells be embryonicstem cells. In another embodiment, it is preferred that they betissue-specific stem cells. In a further embodiment, it is preferredthat the avian embryo cells be somatic tissue-specific stem cells,including hematopoietic stem cells, neural crest cells, and primordialgerm cells.

[0037] The process of gene targeting is dependent on the ability to growembryo cells in culture, which allows time for in vitro genemanipulation, as well as the viability of embryo cells containing theforeign DNA when placed into recipient birds. In avian species, certaindonor cell types have been isolated that retain viability when injectedinto recipient embryos. See Etches et al., in Avian Incubation, Chapter22, Butterworth Publishers (1990); Verrinder Gebbins et al., FourthWorld Congress on Genetics Applied to Livestock Production, Edinburgh,(1990); Petitte et al., Development 108, 185-189 (1990)). These studiesshowed that blastodermal cells derived from Stage X embryos (embryo athatch) remained viable when transferred to comparable recipient Stage Xembryos.

[0038] Embryonic stem cells have been used effectively as cellularvehicles in mice as a means to produce any desired genotype (Capecchi,TIG, 5, 70-76 (1989)). A major advantage of using cells as vehicles forgene transfer is that the incorporation and function of the gene can beevaluated in vitro without screening vast numbers of animals. Inaddition, gene transfer in birds is most likely to be of value to thepoultry industry if the modifications of the genome occur in a specific,site directed manner rather than by a random approach. Targeting ofintroduced genes to specific sites in the chromosome can be achievedusing gene constructs that are capable of undergoing homologousrecombination, in which exogenous and native DNA molecules recombinewithin regions of homology. The attraction of this approach lies in itspotential for modification of endogenous genes in situ to enhance oreliminate expression, to alter tissue specificity, or to alterdevelopmentally regulated expression.

[0039] Another donor cell type that can be effectively transferred isthe primordial germ cell. Such cells can be isolated from the embryonicblood of Stage XVI embryos (55-60 hours of incubation). This is becausethese cells originate outside the embryo in the germinal crescent andmigrate via the blood to the germinal ridge, which is the future site ofthe gonad. Simkiss et al., Protoplasma 151, 164-166 (1989) havedemonstrated that primordial germ cells containing endogenous retroviralmolecules can be transferred to comparable recipient Stage XVI embryosthat lack this marker. The introduction of foreign DNA throughprimordial germ cells can lead to alteration of a number of phenotypicexpressions, including gender.

[0040] Further, phenotype can be altered by the introduction of somatictissue-specific stem cells. In particular, hematopoietic stem cellsintroduced in ovo can colonize the bone marrow and thereafter migrateinto the peripheral blood of the embryo. Such colonization ensureslong-term expression of the transferred gene. As such, this can be aneffective method of increasing the plasma level of specific substances,such as growth factors, immunogens, and the like, in the bloodstream.,and thereby alter the phenotype of the bird.

[0041] While the inventors do not wish to be bound by any theory offeredto explain the mechanism underlying the invention, it is believed thatmigration of cells are usually associated with very primitive cell typessuch as hematopoietic stem cells and primordial germ cells. Both ofthese cell types originate in embryos at sites removed from theirultimate location, and migrate to the aorta, bone marrow, spleen andbursa, and the germinal ridge, respectively, at some point duringembryonic development. Recent evidence in non-avian species suggeststhat the migration exhibited by both hematopoietic stem cells andprimordial germ cells can be attributed to interactions between thec-kit receptor and its ligand, stem cell factor (also known as Mast Cellgrowth factor or Steel factor). It is possible that interactions betweenavian homologs of the c-kit receptor and stem cell factor areresponsible for the migration and colonization of hematopoietic stemcells described below in the Examples. A similar mechanism would likelypermit the engraftment of other self-renewing precursors of amultipotential cell lineage, particularly if the appropriate stem cellis transferred to the recipient embryo at a stage in development whenthat cell type normally colonizes the target tissue.

[0042] It should be noted that embryo cells, such as hematopoieticcells, that can be cultured for relatively long periods of time areadvantageous in that they can be introduced to the bird at a stage ofincubation where rejection or non-integration with host tissue isunlikely. As noted below, the probability of integration of foreigncells with the host is increased by introducing the cells at the stagein the developmental cycle in which such cells are colonizing targettissue.

[0043] An established avian muscle cell line has been shown suitable forintroducing cloned transgenes into recipient embryonic muscle cells(Antin et al., Devel. Biol 143, 122-129 (1991); Antin and Ordahl, Devel.Biol 143, 111-121 (1991)).

[0044] 2. DNA Constructs

[0045] The DNA molecule introduced in ovo is, in general, a constructcontained within an embryo cell comprising a promoter functional inavian cells and a gene encoding a peptide or protein operably linked tothe promoter. Preferably, the protein or peptide is physiologicallyactive and capable of producing a phenotypic change in the bird. Ingeneral, the DNA construct may be a linear DNA molecule or a moleculecarried by a vector or other suitable carrier such as liposomes, calciumphosphate, or DMSO. Vectors, as discussed below, may be plasmids,viruses (including retroviruses), and phage, whether in native form orderivatives thereof. Preferably, the DNA molecule does not containretroviral DNA portions sufficient for integration of the infecting DNAinto the chromosomal DNA of the host bird.

[0046] Illustrative of genes encoding a protein or peptide are thosewhich encode a protein or peptide selected from the group consisting ofgrowth hormone, thyroid releasing hormone (TRH), lymphokines such asinterferon and interleukin-2, insulin-like growth factor, epidermalgrowth factor, and immunogenic recombinant antigens such as thoseproduced by Marek's, infectious bronchitis, mycoplasma, avian leucosis,reovirus, pox, adenovirus, cryptosporidia, chicken anemia agent,Pasteurella species, avian influenze, Marek's MDX, Gumboro Diseasevirus, Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus(IBDV), Rous sarcoma virus, Escherichia coli, and Eimeria tenella.

[0047] The production of cloned genes, recombinant DNA, vectors,transformed host cells, proteins and protein fragments by geneticengineering is well known. See e.g., U.S. Pat. No. 4,761,371 to Bell etal. at Col. 6 line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clarket al. at Col. 4 line 38 to Col 7 line 6; U.S. Pat. No. 4,912,038 toschilling at Col. 3 line 26 to Col. 14 line 12 (applicants specificallyintend that the disclosure of these and all other patent referencescited herein be incorporated herein by reference). Protocols forrestriction endonuclease digestion, preparation of vectors, DNApurification and other such procedures were essentially as described instandard cloning manuals. See Sambrook et al., Molecular Cloning, aLaboratory Manual, (2d Ed., Cold Spring Harbor Press, New York (1989)).

[0048] A vector is a replicable DNA construct used to either amplifyand/or express DNA encoding the gene of interest. A suitable expressionvector will have controlling elements capable of expressing the clonedcDNA or genomic DNA placed in the correct orientation when the vector isintroduced into the correct host. Such elements typically include butare not limited to a promoter region which interacts specifically withcellular proteins involved in transcription, enhancer elements which canstimulate transcription many-fold from linked heterologous promoters, asplice acceptor and/or donor molecules, and termination andpolyadenylation signals. Also required is a DNA sequence for a ribosomebinding site capable of permitting translation which is operably linkedto the gene to be expressed.

[0049] Recently, a muscle-specific promoter has been isolated which ispositioned upstream of both the skeletal muscle structural gene and theessential proximal promoter element and is operably associated witheach. (Mar and Ordahl, Proc. Natl. Acad. Sci. USA 85, 6404-6408 (1988)).Other exemplary promoters operable in avian cells and embryo cellsinclude promoters associated with the genes expressing skeletal actin,phosphoglycerate kinase (PGK), dihydrofolate reductase (DHFR), andchicken β-globin, promoters for hematopoietic stem cell antigens,promoters operably associated with hematopoietic transcription factors,the promoter for Herpes Virus, thymidine kinase and promoters for virallong-terminus repeats, such as Rous Sarcoma Virus.

[0050] Vectors comprise plasmids, viruses (e.g. adenovirus,cytomegalovirus), phage, and DNA fragments integratable into the hostgenome by recombination. The vector replicates and functionsindependently of the host genome.

[0051] C. Subjects and Time of Administration

[0052] The present invention may be carried out on any avian subject,including, but not limited to, chickens, turkeys, ducks, geese, quail,and pheasant. The embryo cells may be introduced in ovo at any timeduring incubation, the duration of which will vary depending upon thespecies. For example, DNA may be introduced into chicken eggs early inincubation (e.g., between about days 2 and 3 of incubation) or late inincubation (e.g., during the last quarter of incubation; i.e., betweenabout day 16 of incubation and hatch).

[0053] It is preferred that the timing of embryo cell introductioncoincide with the embryonic developmental stage in which the introducedcell colonizes in the the embryo. For example, if a hematopoietic stemcell is introduced to a chick embryo, it is preferred that it beintroduced between about day 15 and day 17 of incubation, as it isduring this stage that the endogenous hematopoietic stem cells of chickembryos typically colonize the bone marrow. By timing introductionthusly, the probability that the foreign cells will colonize also can beimproved. Having colonized and thus being integrated with the hosttarget tissue, the cells maintain their capacity for self-renewal andthe capacity for differentiation into multiple lineages.

[0054] The somatic stem cell or primordial germ cell may be introducedinto any region of the egg, including the air cell, the albumen, thechorio-allantoic membrane, the yolk sac, the yolk, the allantois, theamnion, or directly into the embryonic bird. Preferably, the cell isintroduced into either the yolk sac or the air cell, with the air cellbeing a more preferred introduction site. The inventors havedemonstrated that cells introduced to these sites can migrate to thetarget tissue rather than remaining with the site during development.Moreover, both the yolk sac and the air cell are located near theeggshell and thus are relatively easily accessed by injection apparatuswithout damage to other embryonic structures.

[0055] D. Methods of Introducing DNA into Eggs

[0056] Any suitable means may be used for introducing the DNA in ovo,including in ovo injection, high pressure spray through the egg shell,and ballistic bombardment of the egg with microparticles carrying theDNA construct.

[0057] Where in ovo injection is used the mechanism of injection is notcritical, but it is preferred that the method not unduly damage thetissues and organs of the embryo or the extraembryonic membranessurrounding it so that the treatment will not decrease hatch rate. Asmentioned, preferred injection sites include the yolk sac and the aircell, each of which is located near the eggshell and thus is relativelyeasily reached by injection apparatus without damage to other embryonicstructures and without compromising the protection afforded by theeggshell. A hypodermic syringe fitted with a needle of about 18 to 26gauge is suitable for the purpose. Depending on the precise stage ofdevelopment and position of the embryo, a one-inch needle will terminateeither in the fluid above the chick or in the chick itself. Yolk sacinjection can be achieved by insertion of a needle to a depth of betweenabout ½ and 1½ inches into the [side?] portion of the egg. Air cellinjection can be carried out by injection at a depth of between about ⅛and ½ inches into the large end of the egg. Those skilled in this artwill appreciate that the injection depth can vary depending on thedevelopmental stage of the embryo. A pilot hole may be punched ordrilled through the shell prior to insertion of the needle to preventdamaging or dulling of the needle. If desired, the egg can be sealedwith a substantially bacteria-impermeable sealing material such as waxor the like to prevent subsequent entry of undesirable bacteria.

[0058] It is envisioned that a high speed automated injection system foravian embryos will be particularly suitable for practicing the presentinvention. Numerous such devices are available, exemplary being theEMBREX INOVOJECT™ system (described in U.S. Pat. No. 4,681,063 toHebrank), and U.S. Pat. Nos. 4,040,388, 4,469,047, and 4,593,646 toMiller. The disclosure of these references and all references citedherein are to be incorporated herein by reference. All such devices, asadapted for practicing the present invention, comprise an injectorcontaining the DNA as described herein, with the injector positioned toinject an egg carried by the apparatus with the DNA. In addition, asealing apparatus operatively associated with the injection apparatusmay be provided for sealing the hole in the egg after injection thereof.

[0059] The currently preferred apparatus for practicing the presentinvention is disclosed in U.S. Pat. No. 4,681,063 to Hebrank and U.S.Pat. No. 4,903,625 to Hebrank, the disclosure of which are incorporatedherein by reference. This device comprises an injection apparatus fordelivering fluid substances into a plurality of eggs and suctionapparatus which simultaneously engages and lifts a plurality ofindividual eggs from their upwardly facing portions and cooperates withthe injection means for injecting the eggs while the eggs are engaged bythe suction apparatus. The features of this apparatus may be combinedwith the features of the apparatus described above for practicing thepresent invention. Those skilled in the art will appreciate that thisdevice can be adapted for injection into any portion of the egg byadjusting the penetration depth of the injector.

[0060] The present invention is explained further in the followingnon-limiting examples. In these Examples, “μL” means microliters, “mL”means milliliters, “ng” means nanograms, “μg” means micrograms, “mg”means milligrams, “cc” means cubic centimeters, “mm” means millimeters,“mM” means concentration in millimoles, “SSC” means a solution of 0.15Msodium chloride and 0.015M sodium citrate, “Tris” means a buffersolution of tri(hydroxymethyl)animomethane, “EDTA” means a solution ofethylene diamine tetraacetic acid and “° C.” means degrees Celsius.

EXAMPLE 1 Injection of DNA In Ovo

[0061] Using the Embrex Inovoject™ system described above, gene transferis accomplished by injecting 25, 50, or 100 μg of pmiwZ or pRSV-ADH DNAin 100 μL of phosphate buffered saline (PBS) into the embryo in theregion defined by the amnion at day 18 of incubation. Embryos aresacrificed at 19, 20, or 21 days of incubation and muscle tissue isexamined histologically for construct expression. LacZ expression isexamined in living tissue using a non-toxic fluorescent substrate(ImaGene™, Molecular Probes, Inc.) or in fixed tissue using X-gal (Uenoet al., Develop. Growth and Differ. 30(1), 61-73 (1987)). ADH expressionis examined in fixed tissues using 2-butanol (Ordahl, supra (1986)), asubstrate which is specific for Drosophila ADH and cannot be used byvertebrate ADH. Therefore, endogenous expression is able to bedistinguished from exogenous expression.

[0062] When a construct is expressed, the other injected embryos areallowed to hatch and are raised to 1-2 weeks of age. At various pointsduring this time, the birds are sacrificed and the portion of musclecorresponding to the site of injection and expression in the 19-21 dayembryos is analyzed for bacterial β-galactosidase or Drosophila AHDactivity.

EXAMPLE 2 Introduction of Hematopoietic Progenitor Cells In Ovo

[0063] A DNA-liposome complex consists of 25-100 μg of recombinant DNAas described in Example 1 above and 100 μl Lipofectin™ (Gibco/BRL)formed into liposomes in accordance with known techniques. Aortichematopoietic progenitor cells are cultured from dissociated aorta cellsobtained from embryos at 3 days of incubation in accordance with knowntechniques. These cells are transfected in vitro with DNA-liposomecomplexes in accordance with known techniques and injected into the yolksac or chorio-allantoic membrane of recipient chicken embryos in ovo at2-3 days of incubation with an Inovoject™ injection apparatus (Embrex,Inc., Morrisville, N.C.). These embryos are incubated to hatch and theactivity of the transgene assessed in bone marrow cultures and bloodcells at various intervals post-hatch, utilizing the analyticaltechniques described in Example 1 above.

EXAMPLE 3 Preparation of Female Hematopoietic Stem Cells and Injectioninto Male Embryos

[0064] Female chickens carry one W and one Z sex chromosome, while malechickens carry two Z chromosomes. The W chromosome contains a highlyrepetitive DNA sequence which is not found on any other chromosome. Byusing this W chromosome specific repeat sequnce as a molecular marker,hematopoietic cells from female donor birds can be detected in malerecipient birds by Polymerase Chain Reaction (PCR) techniques performedon peripheral blood samples and by in situ hybridization of W-specificlabelled DNA probes to peripheral blood smears of recipient birds.

[0065] To effect the transfer of stem cells from female to male embryos,first viable 16 day embryos were removed from the eggshell. Embryos weredissected to determine the sex visually, and a small blood sample wasobtained to confirm sex determination by PCR with W-chromosome specificprimers. Male embryos were discarded, and female embryos were used fordonors of hematopoietic cells.

[0066] The tibiotarsuses from both legs of female embryos were removedfrom surrounding muscle tissue, and the ends of the bones were trimmed.A 1 cc syringe containing cold sterile phosphate buffered saline (PBS)was inserted into the end of the bone, and bone marrow was eluted fromthe bone itself by dripping PBS through the lumen of the bone. Marrowfrom each donor was harvested and processed independently to minimizethe chance of contamination. Cell numbers of each marrow sample werecounted in a hemocytometer. The samples were centrifuged and resuspendedin a volume of 100 to 500 μL for delivery to recipient birds.

[0067] A small hole was made in the eggshell housing each recipientbird, and donor cells were delivered either to the yolk sac or to theair cell membrane. Both delivery sites required that a hole be punchedin the eggshell. Delivery to the air cell membrane was accomplished bydripping donor cells in PBS onto the membrane, while delivery to theyolk sac required direct penetration of the yolk sac with a needle.Holes in the eggshell were sealed by plastic wrap anchored by petroleumjelly, and the embryos were allowed to hatch.

[0068] Cells were transferred to 82 day 16 embryos: 41 embryos receiveddonor cells on the air cell membrane, and 41 embryos received donorcells in the yolk sac. Twenty-eight of the 41 embryos receiving cells inthe yolk sac hatched (68% hatch percentage), and 37 of 41 embryosreceiving donor cells on the air cell membrane hatched (90% hatchpercentage).

[0069] One week after injection, blood samples were obtained bypuncturing the frontal sinus of the bird and collecting peripheral bloodin syringes coated with 0.5 M EDTA solution (pH 8.0). Chicks were theneuthanized by CO₂ and the sex of each was determined visually. Bloodsamples from males were then used in the PCR and DNA hybridizationstudies that follow.

EXAMPLE 4 PCR Analysis to Detect the Presence of DNA from Donor Femalesin the Blood of Recipient Males

[0070] A 1 μL sample of recipient blood collected by the procedure ofExample 3 was used in the PCR with primers specific for the W-chromosomespecific repetitive sequence. The PCR analysis was carried out accordingto standard techniques, with a positive PCR signal indicating thatfemale donor cells were present in male recipients. A total of 45 malechicks were analyzed by PCR. The data are shown in Table 1. TABLE 1Detection of W-Chromosome sequence in Male Chicks by PCR Delivery ofcells Sex as determined by PCR # chicks air cell Female Male analyzedmembrane yolk sac Air cell Yolk Air cell Yolk 45 33 12 10 5 23 7

[0071] Of the chicks analyzed, 15 (33%) contained female-specificsequences in peripheral blood; 30 (67%) did not score positive forfemale sequences. Of the 15 positive animals, 10 (67%) received donorcells on the air cell membrane and 5 (33%) received donor cells in theyolk sac. The W-chromosome repeat sequence was detected in 30 percent ofthe male embryos injected into the air cell, and the sequence wasdetected in 42 percent of male embryos injected into the yolk sac. Thesedata indicate that the sequence is capable of persisting in the bird forat least one week, and can migrate from the injection site to theperipheral blood of the bird in that time.

EXAMPLE 5 DNA Hybridization Procedure

[0072] The presence of donor hematopoietic cells in peripheral hardblood was also analyzed in situ by a hybridization assay specific forthe W-chromosome repeat sequence. Blood was drawn from recipient birdsas described in Example 3. Slides were pre-treated by dipping in asolution of 500 μg/mL poly-L-lysine and air-drying. Two and 5 μL samplesof blood were smeared on the slides. Slides were incubated in a solutionof 20 mM Tris pH 7.5, 2 mM CaCl₂ and 0.6/ml mg proteinase K for 15minutes at 37° C., followed by two washes of 10 minutes each at roomtemperature with gentle agitation in 0.2% glycine (w/v) in PBS(−). Cellswere fixed by placing slides in 4% (w/v) paraformaldehyde in PBS(−) for10 minutes at room temperature followed by two 15 minute washes in 5 mMMgCl₂ in PBS(−) at room temperature. Samples were dehydrated by washingin 70% and 95% ethanol for 5 minutes each at room temperature, then wereair-dried. DNA on the slides was denatured by incubation in 70% (v/v)formamide, 2×SSC pH 7.0 at 73° C. for 8 to 10 minutes. Slides were thenimmediately washed in 70% ethanol at −20° C. for 5 minutes, washed againfor five minutes in 95% ethanol at −20° C., and air dried.

[0073] A hybridization solution was prepared containing 50% (v/v)formamide, 2×SSC pH 7.0, 10% (w/v) dextran sulfate, 1% (v/v) Tween-20,100 ng/μL denatured salmon sperm DNA, and 0.5-1.0 ng/μL labelledW-chromosome-specific DNA probe. The W-specific probe was labelled byincorporation of digoxigenin-substituted nucleotides using a commercialkit (Boehringer Mannheim).

[0074] To effect hybridization, the hybridization solution was denaturedat 73° C. for ten minutes, then both the hybridization solution and theslides were incubated at 37° C. for 5 minutes. Twenty μL ofhybridization solution was placed on the slide, covered with asiliconized cover slip, and sealed with rubber cement. The slides werethen incubated overnight at 37° C. in a moist environment. The followingday the coverslips were removed by soaking the slides in 50% (v/v)formamide, 2×SSC pH 7.0 at 45° C. Slides were washed three times for 10minutes each at 45° C. in 50% (v/v) formamide, 2×SSC pH 7.0, then washedthree more times for ten minutes each in the 2×SSC pH 7.0 alone at roomtemperature.

[0075] Hybridization of the detector probe to the W-chromosome wasdetected using a colorimetric assay for an alkalinephosphatase-conjugated antibody specific to digoxigenin according to themanufacturer's directions (Boehringer Mannheim). Cells on the slidesstained purple indicated the presence of female specific DNA sequences.All experiments included positive controls comprising slides of samplesdrawn from female chicks.

EXAMPLE 6 Results of DNA Hybridization Assay

[0076] After hybridization under the procedure described in Example 5,slides were scored for the presence or absence of cells containing theW-chromosome-specific repetitive sequence. Blood smears from sixphenotypically male chicks receiving female donor cells were analyzed.The results are shown in Table 2 which sets forth the data by specimennumber and site of delivery. TABLE 2 Detection of W-Chromosome-specificSequence in Male Chicks negative in situ positive in situ Male by PCRFemale by PCR Female by PCR #6111 air cell #7047 yolk sac #500 air cell#7048 yolk sac #6116 air cell #7046 yolk sac

[0077] Of the six chicks analyzed, five had scored positive for femaleDNA by the PCR analysis. Of these five, three indicated hybridization toW-specific probes in in-situ analysis. Blood smears from female chicksprovided positive controls. Chicks scoring positive for female DNAsequences by PCR analysis, yet negative by in situ analysis mayrepresent animals with a very low percentage of transferred donor cellspresent in peripheral blood. In situ analysis of greater numbers ofcells from these chicks may be required to detect female donor cells.

[0078] These results indicate that transfer of genetically engineeredhematopoietic stem cells in ovo represents a viable method of deliveringforeign genes and the proteins encoded by these genes to birds. The dataindicate that hematopoietic cells delivered to the yolk sac or to theair cell membrane in day 16 of incubation embryos can persist for up toone week post-transfer, and can be detected in the peripheral blood ofpost-hatch chicks. In no instance during the procedures of Examples 3-6were donor cells administered directly into the circulatory systems ofembryos. The delivery of hematopoietic stem cells to the air cell in ovois particularly attractive in that an acceptable level of hatchabilitywas maintained; hatchability of about 90% was observed. The ability todeliver a genetically engineered stem cell in ovo while maintaining goodhatchability makes this a feasible approach to delivery of foreign genesand proteins encoded by those genes to the embryo.

[0079] The foregoing examples are illustrative of the present invention,and are not to be construed as limiting thereof. The invention isdefined by the following claims, with equivalents of the claims to beincluded therein.

That which is claimed is:
 1. A method of altering the phenotype of abird, comprising introducing avian somatic tissue-specific stem cellsinto an egg containing a bird during in ovo incubation, said cellscontaining and capable of expressing at least one DNA molecule in anamount effective to cause a change in the phenotype of the bird.
 2. Amethod according to claim 1, further comprising the step of transfectingsaid somatic tissue-specific stem cells with said DNA molecule prior tosaid introducing step.
 3. A method according to claim 1 wherein saidintroducing step is carried out by injecting said somatictissue-specific stem cells into a compartment of the egg selected fromthe group consisting of the air cell and the yolk sac.
 4. A methodaccording to claim 1 wherein said bird is selected from the groupconsisting of chickens, turkeys, ducks, geese, quail and pheasants.
 5. Amethod according to claim 1 wherein said somatic tissue-specific stemcells are introduced in ovo during about the last quarter of incubation.6. A method according to claim 1 wherein said somatic tissue-specificstem cells are introduced in ovo at a stage of development in which saidstem cells colonize tissue of said bird.
 7. A method according to claim1, wherein said somatic tissue-specific stem cells are hematopoieticstem cells.
 8. A method according to claim 1, wherein said somatictissue-specific stem cells are neural crest cells.
 9. A method accordingto claim 1, wherein said somatic tissue-specific stem cells are coupledwith at least one liposome in a DNA-liposome complex.
 10. A methodaccording to claim 1, further comprising the step of incubating said eggto hatch.
 11. A method according to claim 11, further comprising thestep of raising said bird to at least an age at which said change inphenotype is expressed.
 12. A method of altering the phenotype of a birdcomprising introducing avian embryo cells into the air cell of an eggcontaining a bird during in ovo incubation, said embryo cells containingand capable of expressing at least one DNA molecule in an amounteffective to cause a change in the phenotype of the bird.
 13. A methodaccording to claim 12, further comprising the step of transfecting saidembryo cells with said DNA sequence prior to said introducing step. 14.A method according to claim 12 wherein said bird is selected from thegroup consisting of chickens, turkeys, ducks, geese, quail andpheasants.
 15. A method according to claim 12 wherein said avian cellsare introduced in ovo during about the last quarter of incubation.
 16. Amethod according to claim 12, wherein said embryo cells are introducedin ovo at a stage of development in which said stem cells colonizetissue of said bird.
 17. A method according to claim 12, wherein saidembryo cells are coupled with at least one liposome in a DNA-liposomecomplex.
 18. A method according to claim 12, wherein said embryo cellsare primordial germ cells.
 19. A method according to claim 12, whereinsaid embryo cells are embryonic stem cells.
 20. A method according toclaim 12, further comprising the step of incubating said egg to hatch.21. A method according to claim 20, further comprising the step ofraising said bird to at least an age at which said change in phenotypeis expressed.
 22. A method of altering the phenotype of a birdcomprising introducing avian somatic tissue-specific stem cells to theair cell of an egg containing a bird during in ovo incubation, the aviansomatic tissue-specific stem cells containing and capable of expressingat least one DNA molecule in an amount effective to cause a change inthe phenotype of the bird.
 23. A method according to claim 22, furthercomprising the step of transfecting said somatic tissue-specific stemcells with said DNA molecule prior to said introducing step.
 24. Amethod according to claim 22 wherein said bird is selected from thegroup consisting of chickens, turkeys, ducks, geese, quail andpheasants.
 25. A method according to claim 22 wherein said somatictissue-specific stem cells are introduced in ovo during about the lastquarter of incubation.
 26. A method according to claim 22, wherein saidchange in phenotype comprises an increase in growth rate, feedefficiency, disease resistance.
 27. A method according to claim 22,wherein said embryo cells are introduced in ovo at a stage ofdevelopment in which said stem cells colonize tissue of said bird.
 28. Amethod according to claim 22, wherein said somatic tissue-specific stemcells are hematopoietic stem cells.
 29. A method according to claim 22,wherein said somatic tissue-specific stem cells are neural crest stemcells.
 30. A method according to claim 22, wherein said somatictissue-specific stem cells are coupled with at least one liposome in aDNA-liposome complex.
 31. A method according to claim 22, furthercomprising the step of incubating said egg to hatch.
 32. A methodaccording to claim 32, further comprising the step of raising said birdto at least an age at which said change in phenotype is expressed.